The phosphorylation of proteins by mitotic kinases such as Cdc2 on serine or threonine residues preceding proline (Ser/Thr-Pro) plays an essential role in triggering an organized set of structural modifications that occur during mitosis. However, it is not clear how these phosphorylation events affect the activities of specific mitotic phosphoproteins. During a search for proteins that interact with and inhibit the essential mitotic kinase, NIMA, we have previously isolated a new mitotic regulator, Pin1. Pin1 is a novel and conserved cis/trans peptidyl-prolyl isomerase (PPIase) that is essential specifically for mitotic progression. Recent results from our laboratory have demonstrated that phosphorylation not only alters the prolyl isomerization rate of the Ser/Thr-Pro peptide bond, but also serves as a binding site for Pin1. Pin1 is a sequence-specific and phosphorylation- dependent PPIase that binds and regulates the activities of a conserved set of important mitototic phosphoproteins. Our preliminary results further showed that Pin1 itself is phosphorylated in a cell-cycle regulated manner. Together these results suggest a novel signaling regulatory mechanism: the PPIase Pin1, whose function also is likely regulated by phosphorylation, attacks proteins that have been phosphorylated by Pro-directed kinases, and induces a conformational change to regulate their activity. To further test this potential new mitototic regulatory mechanism, we first plan to perform a detailed structure-function analysis on Pin1 protein using in vitro and in vivo assays, and then to elucidate the molecular mechanism of regulation of mitotic phosphoproteins by Pin1. Next, we will define the role of Pin1 in mitotic progression and identify its physiological substrates using both biochemical and genetic approaches. These studies will allow us to address for the first time whether the PPIase activity is essential for cell survival, and to elucidate the molecular mechanism by which Pin1 regulates mitotic progression. Given the current interest in the role of the cell cycle in carcinogenesis and the ability to kill cells specifically at mitosis by inhibiting the Pin1 function, these studies should help elucidate the molecular mechanisms of cell cycle control an carcinogenesis and may lead eventually to development of new therapeutic reagents for cancer.